In recent years, reactive oxygen species (ROS) have been shown to have critical roles in normal vascular function and the pathogenesis of vascular disease. These molecules have profound effects on vascular smooth muscle cell (VSMC) growth, migration and, as we now show, differentiation. In the previous grant period, we focused on NAD(P)H oxidases (Nox), enzymes that are major sources of 02 ?" in vascular cells. We found that Nox1 is localized to caveolae and mediates growth of VSMCs, while Nox4 is found in focal adhesions and the nucleus. Our preliminary data indicate that Nox4 may be important in post-natal differentiation, stress fiber formation and focal adhesion assembly. These data indicate that the subcellular localization of Nox enzymes may be critical to their function. We now propose to examine in more detail the molecular mechanisms linking Nox4 to the regulation of the actin cytoskeleton and differentiation marker gene expression in adult cells. Our proposal centers on Nox4 regulation of the small molecular weight G protein Rho. In the first specific aim, we will define the molecular mechanisms by which Nox4 regulates Rho expression and activity. We hypothesize that Nox4 induces RhoA transcription, and leads to its activation by promoting its release via a guanine dissociation inhibitor, RhoGDI. In Aim 2, we plan to determine the specific molecular pathways by which Nox4 mediates the expression of differentiation marker genes, F-actin polymerization, stress fiber formation and the conversion of focal complexes to mature focal adhesions. We hypothesize that Nox4 activation of Rho stimulates its effectors, Rho kinase and mDia, leading to actin polymerization, focal adhesion assembly and translocation of smooth muscle-specific transcription factors to the nucleus. The sequential molecular pathways responsible for the link between Nox4 and these physiological endpoints will be investigated in detail. In the final aim, we will test some of these molecular relationships in vivo, using unique mouse models of vascular injury and smooth muscle redifferentiation in which Nox4 is upregulated by genetic manipulation of its binding partner. Delineating the functional consequences of Nox4 activation will lead to a better understanding of the mechanisms controlling vascular smooth muscle function in health and disease.